Biotechnological methods are used to an increasing extent in the production of proteins, peptides, nucleic acids and other biological compounds, for research purposes as well as in order to prepare novel kinds of drugs. Due to its versatility and sensitivity to the compounds, chromatography is often the preferred purification method in this context. The term chromatography embraces a family of closely related separation methods, which are all based on the principle that two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components in the sample.
Interactions between a target compound and metal chelating groups present on the stationary phase are utilised in a chromatographic purification method denoted immobilised metal ion adsorption chromatography (IMAC), also known as metal chelating affinity chromatography (MCAC), which is often used for the purification of proteins. The principle behind IMAC lies in the fact that many transition metal ions can coordinate to phosphate groups and nitrogen atoms, such as in the amino acids histidine, cystein, and tryptophan, via electron donor groups on the amino acid side chains. To utilise this interaction for chromatographic purposes, the metal ion must be immobilised onto an insoluble support. This can be done by attaching a chelating group to the chromatographic matrix. Most importantly, to be useful, the metal of choice must have a higher affinity for the matrix than for the compounds to be purified. Examples of suitable coordinating ions are Cu(II), Zn(II), Ni(II), Ca(II), Co(II), Mg(II), Fe(III), Al(III), Ga(IIl), Sc(III) etc.
Various chelating groups are known for use in IMAC, such as iminodiacetic acid (IDA), which is a tridentate chelator, and nitrilotriacetic acid (NTA), which is a tetradentate chelator. Elution of an IMAC resin is as regards proteins commonly performed by addition of imidazol. Alternatively, elution is conventionally performed by lowering the pH.
In recent years, IMAC has successfully been used for the purification of proteins and peptides, wherein His-tags have been introduced by recombinant techniques to facilitate efficient purification thereof by IMAC. For this reason, IMAC has assumed a more important role in large-scale protein and/or peptide production. In addition, IMAC has also been used in purification of phosphorylated proteins and peptides from tryptic protein digests. Such phosphorylated proteins and peptides can subsequently be analysed by ESI/MS/MS to determine the phosphorylated sites therein.
Further, during the period when the IMAC was relatively new, use thereof for purification of various compounds were suggested. For example, Porath et al (U.S. Pat. No. 4,677,027) disclosed in 1985 how biological macromolecules and particles can be separated using a product consisting of a solid phase having immobilised metal ions on its surface substituted via a metal chelate bond with a polymer. The envisaged biomolecules are virus and cells, but polysaccharides, proteins and also oligonucleotides are mentioned. However, since then, oligonucleotides have due to more recent scientific findings found new applications, in turn necessitating novel modifications thereof.
One example of a more recently developed field, wherein oligonucleotides are modified, is the antisense technology in drug discovery. Antisense drugs work at the genetic level to interrupt the process by which disease-causing proteins are produced. This is possible, since proteins have been shown to play a central role in virtually every aspect of human metabolism. Almost all human diseases are the result of inappropriate protein production, or a disordered protein performance. This is true of both host diseases, such as cancer, and infectious diseases, such as AIDS. Traditional drugs are designed to interact throughout the body with protein molecules that support or cause diseases. Antisense drugs are designed to inhibit the production of disease-causing proteins. They can be designed to treat a wide range of diseases including infectious, inflammatory and cardiovascular diseases and cancer and have the potential to be more selective, and, as a result, more effective and less toxic than traditional drugs. The mechanisms behind antisense technology have been widely described, see e.g. Uhlmann et al in Antisense Oligonucleotides: A New Therapeutic Principle, Chemical Reviews, Vol. 90, Number 4, June 1990. In brief, as is well known, during transcription of DNA into RNA, the two complementary strands of the DNA partly uncoil, whereby the strand known as the sense strand separates from the strand known as the antisense strand. The antisense strand is then used as a template for transcribing enzymes that assemble mRNA in the process known as transcription. The mRNA then migrates into the cell, where ribosomes read the encoded information and string together amino acids to form a specific protein in the process known as translation. Now, the antisense drugs are complementary strands of small segments of mRNA, and they can be either DNA or RNA. To create antisense drugs, nucleotides are linked together in short chains known as oligonucleotides. Each antisense drug is designed to bind a specific sequence of nucleotides in its mRNA target to inhibit production of protein encoded by the target mRNA.
The linking together of oligonucleotides can be performed in any kind of commercially available automated solid-phase synthesiser for synthesis of oligonucleotides under cGMP conditions for clinical studies and commercial drug supplies.) In such synthesis, the oligonucleotides, wherein one oxygen atom of the phosphate group of each base in the native nucleic acid has been exchanged for a sulphur atom, are easily produced. However, an inherent problem in the synthesis of such thioated oligonucleotides, herein-denoted antisense oligonucleotides, is the fact that it will be practically impossible to perform with a yield of 100% correctly phosphorothioated oligonucleotides. Instead, a yield in the range of about 70-75% is usually obtained. Accordingly, before any antisense drug can be prepared thereof, the synthesised product will require a subsequent purification in order ensure a sufficient quality.
Reverse phase HPLC is commonly used for purification of antisense oligonucleotides. However, use of high pressures is in general not considered to be advantageous conditions for this kind of process, since it put high demands on the equipment used and also makes the process difficult, and consequently costly, to scale-up. In addition, the organic solvents commonly used in this technology may be undesirable for some applications.
Deshmukh et al (Deshmukh, R. R., Miller, J. E., De Leon, P., Leitch, W. E., Cole, D. L., and Sanghvi, Y. S. in “Process Development for Purification of Therapeutic Antisense Oligonucleotides by Anion-Exchange Chromatography”, Organic Process Research & Development 2000, 4, 205-213) describes the development of an anion-exchange chromatography method for purification of phosphorothioate antisense oligonucleotides. More specifically, 20-mers which are antisense inhibitors of the cell adhesion molecule ICAM-1 were synthesised and subsequently purified on an anion exchanger carrying quaternary arnmonium functional groups on a polystyrene-based matrix (Source 15 and Source Q 30, both from Amersham Biosciences AB, Uppsala, Sweden). The most advantageous resolution is observed for the higher pH value tested for elution, which was pH 11. However, it has still to be shown whether or not a fully thioated 20-mer can be separated from a 20-mer, wherein one or more of the target oxygens have not been substituted with sulphurs. Thus, the selectivity obtainable with ion exchange for purification of antisense oligonucleotides is still not fully satisfactory. In addition, another disadvantage is that such purification of antisense oligonucleotides by anion-exchange chromatography will also require a step of desalting afterwards, which involves a further process step and consequently a higher process cost in total.
Similarly, Deshmukh et al (Deshmukh, R. R., Warner, T. N., Hutchison, F., Murphy, M., Leitch, W. E., De Leon, P., Srivatsa, G. S., Cole, D. L., and Sanghvi, Y. S. in “Large-scale purification of antisense oligonucleotides by high-performance membrane adsorber chromatography”, Journal of Chromatography A, 890 (2000) 179-192) have suggested purification of antisense oligonucleotides using strong anion exchange membranes. However, like in the above described method, the selectivity obtainable is still not fully satisfactory (is this true, can we add any other disadvantages/differences). In addition, use of membranes entails a low capacity and hence large size membranes will be required for a reasonably efficient process. Finally, this method will like the above-discussed anion-exchange also require a step of desalting afterwards.
WO 99/09045 (Somagenics, Inc.) relates to antisense and antigene therapeutics with improved binding properties and methods for their use. More specifically, the invention relates to antisense and antigene oligonucleotides capable of topologically linking to target nucleic acid in a manner that improves translation and transcription inhibitory properties. In one embodiment, phosphorothioate analogues of nucleic acids are disclosed, which have sulphur in place of non-bridging oxygens bonded to phosphorous in terminal or internucleotide phosphates. This modification is allegedly capable of a stronger binding to metallo-affinity chromatography media than the unmodified equivalents. However, there is no suggestion or guidance that metallo-affinity chromatography could be useful to separate oligonucleotides having a varying degree of thioation. Further, in another embodiment, the oligonucleotides have been platinated. Such platinated oligonucleotides are easily separated from reaction mixtures by preparative electrophoresis, or alternatively by ion-exchange column chromatography. It is also suggested to use metallo-affinity chromatography on mercurated columns as a one-step method of purification of platinated oligonucleotides, but this is a mere suggestion. Nothing in this document provides any evidence that such purification would be efficient or even work, and the components of said “reaction mixture” are not defined.
Thus, there is still a need of alternative procedures for the purification of antisense oligonucleotides, especially of methods sensitive enough to separate antisense oligonucleotides of different thioation degree from each other.